This is a Shannon Award providing partial support for the research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon Award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. This proposal is designed to identify physiological mechanisms that regulate stem cell proliferation. We postulated that stromal cell lines in vitro mirror in vivo microenvironmental niches that control stem cells. This premise is supported by our data that certain stromal cell lines in vitro 1) maintain high levels of in vivo reconstituting stem cells with full repopulation capacity; 2) support expansion of primitive stem cells; 3) selectively maintain stem cells that differ in primitiveness. Thus, stromal cell lines are powerful tools for the analysis of the molecules that regulate stem cells and for the characterization of stem cells that differ in primitiveness. We know that stem cells in these cultures are dependent on direct contact to stromal cells and that both adhesion molecules and cytokines are important. We now hypothesize that these stromal cell molecules regulate stem cells via signals that induce proliferation and signals that protect stem cells from differentiation or death. This hypothesis predicts that stem cell maintenance is a function of two, perhaps independent, events: proliferation and persistence. To test this, we propose three interrelated specific aims: 1) How do stromal cell derived cytokines and adhesion molecules control support of stem cells that differ in repopulation capacity? 2) What are the mechanisms by which stromal cells regulate stem cells? 3) Do stem cells of distinct primitiveness differ in phenotype? We shall address these questions by extending our analysis of known and novel stromal cell derived molecules that regulate stem cells, using blocking antibodies and genetically engineered stromal cell lines. To investigate the mechanisms by which these molecules regulate stem cells, we shall assess the kinetics of stem cell proliferation and persistence, the proliferative capacity of stem cell clones, and the cell cycle status of stem cells in response to stromal cell lines. Stem cells that differ in primitiveness will be cultured on different stromal cell lines to assess whether these lines induce or protect stem cells from differentiation. Finally, we propose to identify unique cell surface markers on stromal cell lines that differ in stem cell support and also on stem cells with different repopulation capacity, using a panel of mAb that we have derived previously. These mAbs will aid in the characterization of potential novel effector molecules important for stem cells and will permit the isolation of stem cells with short- and long- term repopulation capacity from freshly explanted marrow. Thus, the proposed studies will define more precisely the mechanisms and the signals important for stem cell proliferation and persistence. Undoubtedly, an understanding of these events will be beneficial for the treatment of a variety of hematopoietic diseases in man, and will be important for stem cell transplantation and gene therapy.